1. Field of the Invention
This invention relates generally to radioimmunoassay and specifically to solid phase radioimmunoassay of digoxin.
Radioimmunoassay (RIA) is a term used to describe any of several methods for determining very low concentrations of substances, which methods are based on the use of radioactively labelled substances which can form immunochemical complexes. The RIA of a substance for which there exists antibodies is based on the observation that an unknown amount of that substance will tend to compete equally with a known amount of that substance (radioactively labelled) for a limited number of complexing sites on antibodies to the substance. Thus, by permitting the above reaction, and separating the immunochemical complexes formed thereby from the reaction solution, the unknown concentration can be determined by relating the radioactivity count of the separated products or remaining solution to a standard curve prepared beforehand using known amounts of labelled and unlabelled substances.
An essential step in RIA is the separation of the immunochemical complexes from the reaction solution. To facilitate this step, various techniques have been devised to immobilize anti-substance antibodies on essentially water-insoluble carrier materials so that a relatively rapid separation can be accomplished with readily available equipment (e.g. a centrifuge). Methods for immobilizing antibodies on such carriers can be found in U.S. Pat. No. 3,555,143, (organic carriers) issued to Axen et al. on Jan. 12, 1971 and U.S. Pat. No. 3,652,761, (inorganic carriers) issued to H. H. Weetall on Mar. 28, 1972. When such carrier materials are used as supports for antibodies in RIA, it has become a common practice to refer to the technique as solid-phase RIA or, simply, SPRIA. The present invention discloses a novel SPRIA for digoxin.
Digoxin is a cardiac glycoside commonly used in very small quantities as a heart stimulant. The difference between therapeutic and toxic amounts of digoxin is often very slight. Thus, it is very important to have methods for accurately determining very small concentrations of digoxin in serum or plasma samples. Since the clinically significant concentration range of digoxin is within the broad range of about 0.5ng to about 10 ng per ml, RIA offers the only practical method for determining digoxin concentrations.
2. Description of Prior Art
Although conventional RIA techniques are known for measuring digoxin concentrations, those techniques are often time-consuming because they require relatively long periods of time for complete separation of digoxin-anti-digoxin antibody complexes. Hence, because of the importance knowing digoxin concentrations as rapidly as possible, attention has been made in recent years to develop a SPRIA for digoxin which would permit rapid separation and accuracy within the clinically significant concentration range. Methods for conjugating digoxin residues to the amino groups of lysine residues in human serum albumin are disclosed by T. W. Smith et al. in Biochemistry, 9, No. 2, 331-337 (1970) and by V. P. Bulter et al. in Proc. N.A.S., 57, 71-78 (1966). A method of labelling a digoxin derivative with .sup.125 I is disclosed by Gutcho et al. in Clin. Chem. 19/9, 1058-59 (1973). As mentioned above, various techniques for coupling antibodies to inorganics through silanes are disclosed in U.S. Pat. No. 3,652,761, issued to H. H. Weetall. In those disclosures which disclose a specific SPRIA for digoxin, however, the methods of separating immunochemical complexes generally involve using a centrifuge. Although the use of a centrifuge facilitates separation and hastens obtaining the assay results, the use of a centrifuge does not readily permit instrumentation of the SPRIA of digoxin. Hence, there has been a need for another method of separating the reaction products of SPRIA which method could be readily instrumentized for quicker assay results.
In an article by P. J. Robinson et al in Biotech. Bioeng. XV, 603-606 (1973) there are suggested various techniques for separating immobilized enzymes by coupling the enzymes to magnetically responsive inorganic particles and then using magnetism to effect the separation. We are unaware, however, of the use of a similar technique to provide for magnetic separations in a SPRIA of digoxin, especially in a SPRIA of digoxin which can be successfully used to measure clinically significant amounts of digoxin. We have found that a successful SPRIA of digoxin is possible using magnetically responsive carrier materials. The SPRIA lends itself to instrumentation. Details of our composites and methods of making and using them are described below.